Compounds that suppress cancer cells and exhibit antitumor activity

ABSTRACT

The present invention provides compounds S3I-201.1066 (Formula 1) and S3I-201.2096 (Formula 2) as selective Stat3 binding agents that block Stat3 association with cognate receptor pTyr motifs, Stat3 phosphorylation and nuclear translocation, Stat3 transcriptional function, and consequently induced Stat3-specific antitumor cell effects in vitro and antitumor response in vivo.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a National Phase Application of InternationalApplication No. PCT/US2010/001021 filed Apr. 5, 2010, which claimspriority to U.S. Provisional Patent Application No. 61/246,695 filedSep. 29, 2009 and to U.S. Provisional Patent Application No. 61/166,865filed Apr. 6, 2009, each of which is incorporated in its entirety bythis reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. R01CA106439 and R01 CA128865 awarded by the National Institutes of Health.The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of drug development and, moreparticularly, to compounds that inhibit cancer cells.

BACKGROUND OF THE INVENTION

Signal transduction proteins have increased importance in carcinogenesisand tumor formation and represent attractive targets for the developmentof novel anticancer therapeutics.

The Signal Transducer and Activator of Transcription (STAT) family ofproteins are cytoplasmic transcription factors with important roles inmediating responses to cytokines and growth factors, including promotingcell growth and differentiation, and inflammation and immune responses(1,2). Normal STATs activation is initiated by the phosphorylation of acritical tyrosine residue upon the binding of cytokines or growthfactors to their cognate receptors. The phosphorylation is induced bygrowth factor receptor tyrosine kinases, or cytoplasmic tyrosinekinases, including Janus kinases or the Src family kinases. Whilepre-existing dimers have been detected (3,4), phosphorylation isobserved to induce dimerization between two STAT monomers through aphosphotyrosine interaction with the SH2 domain. In the nucleus, activeSTAT dimers bind to specific DNA-response elements in the promoters oftarget genes and regulate gene expression.

It is now well established that aberrant activation of the member of thefamily, Stat3 contributes to malignant transformation and tumorigenesis.Aberrant Stat3-mediated oncogenesis and tumor formation is due in partto the transcriptional upregulation of critical genes, which in turnlead to dysregulation of cell growth and survival, and the promotion ofangiogenesis (2, 5-11) and tumor immune-tolerance (12, 13). Thus,targeting of aberrant Stat3 signaling would provide a novel strategy fortreating the wide variety of human tumors that harbor abnormal Stat3activity.

The critical step of dimerization (14) between two monomers within thecontext of STAT activation presents an attractive strategy to interferewith Stat3 activation and functions and this approach has been exploitedin prior work (15-25). Leading agents from those earlier studies havebeen explored for rational design in conjunction with molecular modelingof the binding to the Stat3 SH2 domain(18, 19), per the X-ray crystalstructure of the Stat3 homodimer (26). One of those leads, S3I-201 (18)had previously been shown to exert antitumor effects against humanbreast cancer xenografts via mechanisms that involve the inhibition ofaberrant Stat3.

In the present study, key structural information from the computationalmodeling of S3I-201 bound to the Stat3 SH2 domain facilitated the designof novel analogs of which S3I-201.1066 and S3-201.2096 show improvedStat3-inhibitory activity. Both S3I-210.1066 and S3I-201.2096 inhibitStat3 activity with IC₅₀ values of 35 and 45 μM, respectively. Thisdisclosure presents evidence that S3I-201.1066 interacts with the Stat3protein and disrupts Stat3 binding to its cognate pTyr peptide ofreceptors. Furthermore, S3I-201.1066 induces antitumor cell effectsselectively in malignant cells harboring aberrant Stat3 and antitumorresponse in vivo in human breast xenografts.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides a useful expansion of several prior studies that have providedthe proof-of-concept for the therapeutic effects of Stat3 inhibitors inhuman tumors. The molecular modeling of the phosphotyrosine (pTyr)-SH2domain interaction in Stat3:Stat3 dimerization, combined with in silicostructural analysis of the previously reported Stat3 dimerizationdisruptor, S3I-201, has furnished a diverse set of analogs.

Herein we disclose that compounds S31201.1066 and S3I-201.2096selectively inhibit Stat3 DNA-binding activity in vitro, with IC₅₀values of 35 and 45 μM, respectively. In vitro biochemical andbiophysical studies show that S3I-201.1066 interacts with Stat3 or theSH2 domain, with an affinity (KD) of 2.74 μM, and disrupts Stat3 bindingto the cognate pTyr-peptide motif, with an IC₅₀ value of 23 μM.Accordingly, S3I-201.1066 blocks Stat3 association with the epidermalgrowth factor (EGF) receptor in EGF-stimulated fibroblasts or in cancercells, consequently inhibiting Stat3 phosphorylation, nucleartranslocation and transcriptional activity, without affecting theErk^(MAPK) pathway.

Furthermore, treatment with S3I-201.1066 selectively suppressed thegrowth, viability, survival and malignant transformation of human breast(MDA-MB-231) and pancreatic (Panc-1) cancer lines and v-Src-transformedmouse fibroblasts harboring aberrant Stat3, and down-regulated theexpression of known Stat3-regulated genes, including c-Myc, Bcl.xL, thematrix metalloproteinase 9, and VEGF. Importantly, treatment withS3I-201.1066 induced strong tumor regression in xenografts of the humanbreast cancer line MDA-MB-231.

Taken together, the present disclosure identifies compounds S3I-201.1066(Formula 1) and S3I-201.2096 (Formula 2) as selective Stat3 bindingagents that block Stat3 association with cognate receptor pTyr motifs,Stat3 phosphorylation and nuclear translocation, Stat3 transcriptionalfunction, and consequently induced Stat3-specific antitumor cell effectsin vitro and antitumor response in vivo.

Accordingly, the present disclosure provides a novel compound, analog ofS31-201, according to Formula 1, as set forth below and in FIG. 1, andsalts thereof.

The disclosure also contemplates that the invention includes thecompound of Formula 1 used in a pharmaceutical composition acceptablefor administration to a patient.

Those skilled in the art should recognize that the compounds of thisinvention may be administered to mammals, preferably humans, eitheralone or in combination with pharmaceutically acceptable carriers,excipients or diluents, in a pharmaceutical composition, according tostandard pharmaceutical practice. The compounds can be administered byany route but are preferably administered parenterally, including byintravenous, intramuscular, intraperitoneal, subcutaneous, rectal andalso by topical routes of administration.

The term “composition” is intended to encompass a product comprising thedisclosed compounds in amounts effective for causing the desired effectin the patient, as well as any product which results, directly orindirectly, from combination of the specific ingredients. However, theskilled should understand that when a composition according to thisinvention is administered to a human subject, the daily dosage of activeagents will normally be determined by the prescribing physician with thedosage generally varying according to the age, weight, sex and responseof the individual patient, as well as the severity of the patient'ssymptoms.

The terms pharmaceutical composition, pharmaceutically and/orpharmacologically acceptable for administration to a patient refer tomolecular entities and/or compositions that do not produce an adverse,allergic and/or other untoward reaction when administered to a subject,be it animal or human, as appropriate.

As known to the skilled, a pharmaceutically acceptable composition orcarrier includes any and/or all solvents, dispersion media, coatings,antibacterial and/or antifungal agents, isotonic and/or absorptiondelaying agents and/or the like. The use of such media and/or agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media and/or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. For administration, preparations should meetsterility, pyrogenicity, general safety and/or purity standards asrequired by FDA Office of Biologics standards.

The skilled will find additional guidance in preparation ofpharmaceutically acceptable compositions by consulting United StatesPharmacopeia (USP) or other similar treatises employed in thepharmaceutical industry.

The novel compound of Formula 1, given above, may be used in variousmethods of treatment, for example: a method of treatment effective toinhibit a cancer cell by contacting the cell with said compound; amethod of treatment effective to inhibit a human pancreatic cancer cellby contacting the cell with said compound; a method of treatmenteffective to inhibit a human breast cancer cell by contacting the cellwith said compound; a method of treatment effective to inhibit a cellcharacterized by an aberrant level of Stat3 by contacting the cell withsaid compound; to inhibit a cell characterized by an aberrant level ofStat3 by contacting the cell with said compound so as to selectivelybind Stat3; to down-regulate expression of Stat3-regulated genes in acell by contacting the cell with said compound; to selectively inhibitStat3-DNA binding activity in a cell by contacting the cell with saidcompound; to block Stat3 association with epidermal growth factorreceptor in EGF-stimulated fibroblasts by contacting the fibroblastswith said compound; and to inhibit tumor cells dependent on aberrantStat3-mediated oncogenesis by contacting the tumor cells with saidcompound so as to interfere with Stat3 function.

The present disclosure also contemplates a second compound, related tothe compound of Formula 1 by both being analogs of S3I-201. This secondcompound is shown below according to Formula 2 and salts thereof.

The compound of Formula 2 has properties that parallel those of thecompound of Formula 1 and may be employed in a likewise manner, asdescribed above.

As used herein, the terms “treat,” “treating” or “method of treatment”refer to both therapeutic treatment and prophylactic or preventivemeasures, wherein the object is to prevent or slow down (lessen) anundesired physiological change or disorder, such as the development orspread of cancer or other proliferation disorder. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. For example, treatment with a compound of the inventionmay include reduction of undesirable cell proliferation, and/orinduction of apoptosis and cytotoxicity.

“Treatment” can also mean prolonging survival as compared to expectedsurvival if not receiving treatment. Those in need of treatment includethose already with the condition or disorder as well as those prone tohave the condition or disorder or those in which the condition ordisorder is to be prevented or onset delayed. Optionally, the patientmay be identified (e.g., diagnosed) as one suffering from the disease orcondition (e.g., proliferation disorder) prior to administration of theStat3 inhibitor of the invention.

The terms “effective to inhibit” or “growth inhibitory amount” of thecompounds of the invention refer to an amount which reduces (i.e., slowsto some extent and preferably stops) proliferation of a target cell,such as a tumor cell, either in vitro or in vivo, irrespective of themechanism by which cell growth is inhibited (e.g., by cytostaticproperties, cytotoxic properties, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings,presented for solely for exemplary purposes and not with intent to limitthe invention thereto, and in which:

FIG. 1(A-C), illustrates structures of (A) S3I-201, (B) S3I-201.1066 and(C) S3I-201.2096; D and E, GOLD docking of (D) S3I-201 (green), and (E)S3I-201.1066 (yellow) and S3I-201.2096 (green) to the SH2 domain ofStat3; arrow denotes potential binding sub-pocket;

FIG. 2 shows effects of S3I-201.1066 and S3I-201.2096 on the activitiesof STATs, Src, Jak1, Shc, and Erks. (A) Nuclear extracts of equal totalprotein containing activated Stat1, Stat3, and/or Stat5 werepre-incubated with or without (i), (iii) and (iv) S3I-201.1066 or (ii)S3I-201.2096 for 30 min at room temperature prior to the incubation withthe radiolabeled hSIE probe that binds Stat1 and Stat3 or the MGFe probethat binds Stat5 and subjecting to EMSA analysis; (B) Nuclear extractsof equal total protein prepared from malignant cells following 24-htreatment with or without S3I-201.1066 were subjected to in vitroDNA-binding assay using the radiolabeled hSIE probe and analyzed byEMSA; (C) Cytosolic extracts of equal total protein were prepared from36-h S3I-201.1066-treated or untreated NIH3T3/v-Src fibroblasts thatstably express the Stat3-dependent luciferase reporter (pLucTKS3) andanalyzed for luciferase activity using a luminometer; and (D) SDS-PAGEand Western blotting analysis of whole-cell lysates of equal totalprotein prepared from S3I-201.1066-treated or untreated NIH3T3/v-Src andPanc-1 cells probing for pY705Stat3, Stat3, pY416Src, Src, pJak1, Jak1,pShc, Shc, pErk1/2 and Erk1/2. Positions of STATs:DNA complexes orproteins in gel are labeled; control lanes (0) represent nuclearextracts treated with 0.05% DMSO, or nuclear extracts or whole-celllysates prepared from 0.05% DMSO-treated cells. Data are representativeof 3-4 independent determinations. ** −<0.01;

FIG. 3 depicts studies of the interaction of S3I-201.1066 with Stat3 orthe Stat3 SH2 domain. (A) EMSA analysis of in vitro binding of Stat3 tothe radiolabeled hSIE probe using nuclear extracts containing activatedStat3 pre-incubated with 0-100 μM S3I-201.1066 in the presence orabsence of increasing concentrations of purified His-tagged Stat3 SH2domain; (B) Surface Plasmon Resonance analysis of the binding of (i)GYLPQTV-NH2 (unphosphorylated, high affinity gp-130 peptide), (ii)GpYLTQTV-NH2 (phosphorylated), or (iii) small-molecule S3I-201.1066 asanalyte to the purified His-tagged Stat3 immobilized on HisCap SensorChip; and (C) Fluorescence Polarization assay of the binding to the5carboxyfluorescein-GpYLPQTV-NH2 probe of (i) increasing concentrationof purified His-Stat3 or (ii) a fixed amount of purified His-Stat3 (150nM) in the presence of increasing concentrations of S3I201.1066.Stat3:DNA complexes in gel are shown, control (-) lane or zero (0)represent 0.05% DMSO. Data are representative of 2-4 independentdeterminations;

FIG. 4 shows the effect of S3I-201.1066 on the colocalization orassociation of Stat3 with EGF receptor and on Stat3 nucleartranslocation. (A) Immunofluorescence imaging/confocal microscopy ofStat3 colocalization with EGFR and Stat3 nuclear localization inEGF-stimulated (1 g/ml; 10 min) NIH3T3/hEGFR pre-treated with or without50 μM S3I-201.1066 for 30 min; or (B) Immunoblotting analysis of (i)EGFR immunecomplex (upper panel) or whole-cell lysates (lower panel)from S3I201.1066-treated Panc-1 and MDA-MB-231 cells, or (ii)immunecomplexes of EGFR (upper panel) or Stat3 (lower panel) treatedwith the indicated concentrations of S3I-201.1066 and probing for EGFR,Stat3, Shc, Grb 2, or Erk1/2MAPK. Data are representative of threeindependent studies;

FIG. 5 indicates that S3I-201.1066 suppresses viability, growth andsurvival of malignant cells that harbor persistently-active Stat3 Humanbreast (MDA-MB-231) and pancreatic (Panc-1) cancer cells, the normalmouse fibroblasts (NIH3T3) and their v-Src transformed (NIH3T3/v-Src) orv-Ras transformed (NIH3T3/v-Ras) counterparts, mouse thymic epithelialstromal cells (TE-71), Stat3 null mouse embryonic fibroblasts(Stat3−/−), and the normal human pancreatic duct epithelial cells(HPDEC) were treated once or untreated with 30-100 M S3I-201.1066 for24-144 h. Cells were (A) assayed for viability using CyQuant cellproliferation kit; IC₅₀ values were derived from graphicalrepresentation; (B) harvested at each 24-h period following treatmentand viable cells counted by trypan blue exclusion with phase-contrastmicroscopy; or (C) allowed to culture until large colonies were visible,which were stained with crystal violet and enumerated. Values are themean and S.D. of 3-4 independent determinations. p values, * −<0.05, and** −<0.01;

FIG. 6 demonstrates that S3I-201.1066 blocks Stat3-dependent malignanttransformation and inhibits the migration of malignant cells harboringaberrant Stat3. (A) Viral Src-transformed mouse fibroblasts(NIH3T3/v-Src) and counterpart transformed by v-Ras (NIH3T3/v-Ras)growing in soft-agar suspension were treated with or without 30-100 μMS3I-201.1066 every 2-3 days until large colonies were visible, whichwere stained with crystal violet and enumerated; (B) Wound healing assayfor effect on cell migration in which human breast (MDA-MB-231) andpancreatic (Panc-1) cancer cells, and the v-Src transformed mousefibroblasts (NIH3T3/v-Src) and counterparts transformed by v-Ras(NIH3T3/v-Ras) were treated with or without 30-80 μM S3I-201.1066 for12-24 h and allowed to migrate into the denuded area. Cell migration wasvisualized at 10× magnification by light microscopy and cells thatmigrated into the denuded area counted and plotted against theconcentration of S3I-201.1066. Values are the mean and S.D. of 3independent determinations. p values, * −<0.05, and ** −<0.01.

FIG. 7 shows that S3I-201.1066 suppresses c-Myc, Bcl-xL, MMP-9 and VEGFexpression. SDS-PAGE and Western blotting analysis of whole-cell lysatesprepared from the human breast cancer MDA-MB-231 and pancreatic cancerPanc-1 cells untreated (DMSO, control) or treated with 80-100 μMS3I-201.1066 for 48 h and probing with anti-Myc, Bcl-xL, MMP-9, VEGF orβ-actin antibodies. Positions of proteins in gel are shown. Data arerepresentative of 3 independent determinations; and

FIG. 8 shows that S3I-M2001 inhibits growth of human breast tumorxenografts. Human breast (MDA-MB231) tumor-bearing mice were givenS3I-201.1066 (3 mg kg-1) or vehicle (0.1% DMSO in PBS) i.v. every 2 or 3days. Tumor sizes, measured every 2 or 3 days, were converted to tumorvolumes and plotted against treatment days; Values are the mean and S.D.from replicates of 12 tumor-bearing mice in each group. *<0.05.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, or other referencesmentioned or cited herein are incorporated by reference in theirentirety. In case of conflict, the present specification, including anydefinitions, will control. In addition, those of skill in the art shouldrecognize that the materials, methods and examples given areillustrative in nature only and not intended to be limiting.Accordingly, this invention may be embodied in many different forms andshould not be construed as limited to the illustrated embodiments setforth herein. Rather, the illustrated embodiments are provided so thatthis disclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art. Other features andadvantages of the invention will be apparent from the following detaileddescription, and from the claims.

The abbreviations used herein are: STAT, signal transducer and activatorof transcription; PBST, phosphate-buffered saline Tween-20®; HPDEC,normal human pancreatic duct epithelial cell line; Stat3−/−, Stat3knockout mouse embryonic fibroblasts; PBS, phosphate-buffered saline;

EMSA, electrophoretic mobility shift assay; Erk, extracellularsignal-regulated kinase; FBS, fetal bovine serum, MMP-9, matrixmetalloproteinases 9.

Experimental Procedures

Cells and Reagents

Normal mouse fibroblasts (NIH3T3) and counterparts transformed by v-Src(NIH3T3/v-Src), v-Ras (NIH3T3/v-Ras) or overexpressing the humanepidermal growth factor (EGF) receptor (NIH3T3/hEGFR), and the humanbreast cancer (MDA-MB-231) and pancreatic cancer (Panc-1) cells have allbeen previously reported (15, 27-29). The normal human pancreatic ductepithelial cells (HPDEC) was a kind gift from Dr. Tsao, (OCI, UHN-PMH,Toronto) (30), Stat3 knockout mouse embryonic fibroblasts was generouslyprovided by Dr. Poli (31), and the mouse thymic epithelial stromal cells(TE-71) was a generous gift from Dr. Farr (32). The Stat3dependentreporter, pLucTKS3 and the v-Src transformed mouse fibroblasts thatstably express pLucTKS3 have been previously reported (15, 33). Cellswere grown in Dulbecco's modified Eagle's medium (DMEM) containing 10%heat-inactivated fetal bovine serum, or in the case of HPDEC, they weregrown in Keratinocyte-SFM media supplemented with 0.2 ng EGF, 30 μg/mlbovine pituitary extract and containing antimycol. Antibodies againstStat3, pY705Stat3, Src, pY416Src, Jak1, pJak1, Shc, pShc, Erk1/2, andpErk1/2 are from Cell Signaling Technology (Danvers, Mass.).

Cloning and Protein Expression

Coding regions for the murine Stat3 protein and Stat3 SH2 domain wereamplified by PCR and cloned into vectors pET-44 Ek/LIC (Novagen) and pETSUMO (Invitrogen), respectively.

The primers used for amplification were: Stat3 Forward:GACGACGACAAGATGGCTCAGTGGAACCAGCTGC (SEQ ID NO:1); Stat3 Reverse:GAGGAGAAGCCCGGTTATCACATGGGGGAGGTAGCACACT (SEQ ID NO:2); Stat3-SH2Forward: ATGGGTTTCATCAGCAAGGA (SEQ ID NO:3); Stat3-SH2 Reverse:TCACCTACAGTACTTTCCAAATGC (SEQ ID NO:4).

Clones were sequenced to verify the correct sequences and orientation.His-tagged recombinant proteins were expressed in BL21(DE3) cells, andpurified on Ni-ion sepharose column.

Nuclear Extract Preparation, Gel Shift Assays, and DensitometricAnalysis

Nuclear extract preparations and electrophoretic mobility shift assay(EMSA) were carried out as previously described (28,33). The ³²P-labeledoligonucleotide probes used were hSIE (high affinity sis-inducibleelement from the c-fos gene, m67 variant, 5′AGCTTCATTTCCCGTAAATCCCTA;SEQ ID NO:5) that binds Stat1 and Stat3 (34) and MGFe (mammary glandfactor element from the bovine β-casein gene promoter,5′-AGATTTCTAGGAATTCAA; SEQ ID NO:6) for Stat1 and Stat5 binding (35,36).Except where indicated, nuclear extracts were pre-incubated withcompound for 30 min at room temperature prior to incubation with theradiolabeled probe for 30 min at 30° C. before subjecting to EMSAanalysis. Bands corresponding to DNA-binding activities were scanned andquantified for each concentration of compound using ImageQuant andplotted as percent of control (vehicle) against concentration ofcompound, from which the IC₅₀ values were derived, as previouslyreported (37).

Immunoprecipitation and SDS-PAGE/Western Blotting Analysis

Immunoprecipitation from whole-cell lysates and SDS/PAGE and Westernblotting analysis were performed as previously described (33,38).Primary antibodies used were anti-Stat3, pY705Stat3, pY416Src, Src,pErk1/2, Erk1/2, pJak1, Jak1, pShc, Shc, Grb 2, c-Myc, Bcl-xL, MMP-9,and β-Actin (Cell Signaling, Danvers), and VEGF (Santa Cruz Biotech,Santa Cruz).

Cell Viability and Proliferation Assay

Cells in culture in 6-well or 96-well plates were treated with orwithout

S3I-201.1066 for 24-144 h and subjected to CyQuant cell proliferationassay (Invitrogen Corp/Life Technologies Corp, Carlsbad, Calif.), orharvested, and the viable cells counted by trypan blue exclusion withphase contrast microscopy.

Immunofluorescence Imaging/Confocal Microscopy

NIH3T3/hEGFR cells were grown in multi-cell plates, serum-starved for 8h and treated with or without S3I-201.1066 for 30 min prior tostimulation by rhEGF (1 g/ml) for 10 min. Cells were fixed with ice-coldmethanol for 15 min, washed 3 times in PBS, permeabilized with 0.2%Triton® X-100 for 10 min, and further washed 3-4 times with PBS.Specimens were then blocked in 1% BSA for 30 min and incubated with EGFR(Santa Cruz) or Stat3 (Cell Signaling Tech) antibody at 1:50 dilution at4° C. overnight. Subsequently, cells were rinsed 4-5 times in PBS,incubated with Alexafluor 546 rat antibody for EGFR detection and Alexafluor 488 rabbit antibody for Stat3 detection (Invitrogen) for 1 h atroom temperature in the dark. Specimens were then washed 5 times withPBS, covered with cover slides with VECTASHIELD mounting mediumcontaining DAPI, and examined immediately under a Leica TCS SP5 confocalmicroscope (Germany) at appropriate wavelengths. Images were capturedand processed using the Leica TCS SP 5 software.

Soft-Agar Colony Formation Assay

Colony formation assays were carried out in 6-well dishes, as describedpreviously (16,37). Briefly, each well contained 1.5 ml of 1% agarose inDulbeco's modified Eagle's medium as the bottom layer and 1.5 ml of 0.5%agarose in Dulbeco's modified Eagle's medium containing 4000 or 6000NIH3T3/v-Src or NIH3T3/v-Ras fibroblasts, respectively, as the toplayer. Treatment with S3I201.1066 was initiated 1 day after seedingcells by adding 80 μl of medium with or without S31201.1066, andrepeating every 3 days, until large colonies were evident. Colonies werequantified by staining with 20 p1 of 1 mg/ml crystal violet, incubatingat 37° C. overnight, and counting the next day under phase contrastmicroscope.

Fluorescence Polarization Assay

Fluorescence Polarization (FP) Assay was conducted as previouslyreported (23), with some modification using the phospho-peptide,5-carboxyfluoresceinGpYLPQTV-NH2 (where pY represents phospho-Tyr) asprobe and Stat3. The FP assay was used to evaluate the binding of agentsto Stat3 and to determine the ability to disrupt the Stat3:pTyr peptideinteraction. A fixed concentration of the fluorescently-labeled peptideprobe (10 nM) was incubated with increasing concentration of the Stat3protein for 30 min at room temperature in the buffer, 50 mM NaCl, 10 mMHEPES, 1 mM EDTA, 0.1% Nonidet P-40, and the fluorescent polarizationmeasurements were determined using the POLARstar Omega (BMG LABTECH,Durham, N.C.), with the set gain adjustment at 35 mP. The Z′ value wasderived per the equationZ′=1−(3SD_(bound)+3SD_(free))/(mP_(bound)−mP_(free)), where SD is thestandard deviation and mP is the average of fluorescence polarization.In the bound state, 10 nM 5-carboxyfluorescein-GpYLPQTV-NH2 wasincubated with 150 nM purified Stat3 protein, while the free (unbound)state was the same mixture, but incubated with an additional 10 μMunlabeled Ac-GpYLPQTV-NH₂. For evaluating agents, Stat3 protein (150 nM)was incubated with serial concentrations of S3I-201.1066 at 30° C. for60 min in the indicated assay buffer conditions. Prior to the additionof the fluorescence probe, the protein:S3I-201.1066 mixtures wereallowed to equilibrate at room temperature for 15 min. Probe was addedat a final concentration of 10 nM and incubated for 30 min at roomtemperature following which the FP measurements were taken using thePOLARstar Omega, with the set gain adjustment at 35 mP.

Surface Plasmon Resonance Analysis

SensiQ and its analysis software Qdat (ICX Technologies, Oklahoma City,Okla.) were used to analyze the interaction between agents and the Stat3protein and to determine the binding affinity. Purified Stat3 wasimmobilized on a HisCap Sensor Chip by injecting 50 g/ml of Stat3 ontothe chip. Various concentrations of S3I-201.1066 in running buffer (1×PBS, 0.5% DMSO) were passed over the sensor chip to produce responsesignals. The association and dissociation rate constants were calculatedusing the Qdat software. The ratio of the association and dissociationrate constants was determined as the affinity (K_(D)).

Colony Survival Assay

This was performed as previously reported (39). Briefly, cells wereseeded as single-cell in 6-cm dishes (500 cells per well), treated oncethe next day with S3I-201.1066 for 48 h, and allowed to grow until largecolonies were visible. Colonies were stained with crystal violet for 4 hand counted under phase-contrast microscope.

Wound Healing Assay for Migration

Wounds were made using pipette tips in monolayer cultures of cells insix-well plates. Cells were treated with or without increasingconcentrations of S3I-201.1066 and allowed to migrate into the denudedarea for 12-24 hours. The migration of cells was visualized at a 10×magnification using an Axiovert 200 Inverted Fluorescence Microscope(Zeiss, Göttingen Germany), with pictures taken using a mounted CanonPowershot A640 digital camera (Canon USA, Lake Success, N.Y.). Cellsthat migrated into the denuded area were quantified. Mice and in vivotumor studies-Six-week-old female athymic nude mice were purchased fromHarlan and maintained in the institutional animal facilities approved bythe American Association for Accreditation of Laboratory Animal Care.Athymic nude mice were injected subcutaneously in the left flank areawith 5×10⁶ human breast cancer MDA-MB-231 cells in 100 μL of PBS. After5 to 10 days, tumors of a diameter of 3 mm were established. Animalswere grouped so that the mean tumor sizes in all groups were nearlyidentical, then given S3I-201.1066 intravenously at 3 mg/kg every 2 orevery 3 days for 17 days and monitored every 2 or 3 days, and tumorsizes were measured with calipers. Tumor volume was calculated accordingto the formula V=0.52×a²×b, where a, smallest superficial diameter, b,largest superficial diameter.

Statistical Analysis

Statistical analysis was performed on mean values using Prism GraphPadSoftware, Inc. (La Jolla, Calif.). The significance of differencesbetween groups was determined by the paired t-test at p<0.05*, <0.01**,and <0.001***.

Results

Computer-Aided Design of S3I-201 Analogs as Stat3 Inhibitors.

Close structural analysis of the lowest Genetic Optimization for LigandDocking (GOLD) (40) conformation of the lead Stat3 inhibitor, S3I-201(yellow) (IC₅₀ =86 μM for inhibition of Stat3:Stat3 (18)) (FIG. 1A)bound within the Stat3 SH2 domain (FIG. 1D), per the X-ray crystalstructure of DNA-bound Stat3β homodimer (26) showed significantcomplementary interactions between the protein surface and the compoundand identified key structural requirements for tight binding. Dockingstudies permitted in silico structural design of analogs of differingStat3 SH2 domain-binding characteristics in order to derive Stat3inhibitors of improved potency and selectivity. GOLD docking studiesshowed limited structural occupation of the Stat3-SH2 domain,identifying a potential means for improving inhibitor potency. The SH2domain is broadly composed of three sub-pockets, only two of which areaccessed by S3I-201 (FIG. 1D). Lead agent, S3I-201 (FIG. 1A) has aglycolic acid scaffold with its carboxylic acid condensed with ahetero-trisubstituted aromatic species to furnish the amide bond, and ahydroxyl moiety that has been tosylated. The ortho-hydroxybenzoic acidcomponent is a known pTyr mimetic, and low energy GOLD docking studiesconsistently placed this unit in the pTyr-binding site, making hydrogenbonds and electrostatic interactions with Lys591, Ser611, Ser613 andArg609. Due to the strength of such interactions between oppositelycharged ions, it is likely that a considerable portion of the bindingbetween the SH2 domain and S3I-201 arises from the pTyr mimetic. TheOtosyl group binds in the mostly-hydrophobic pocket that is derived fromthe tetramethylene portion of the side chain of Lys592 and thetrimethylene portion of the side chain of Arg595, along with Ile597 andIle634. Given the potency of S3I-201 towards Stat3 inhibition, arational synthetic program was undertaken to modify and optimize thecore scaffold to furnish more potent analogs. We additionally exploitedkey hydrophobic interactions with Phe716, 11e659, Val637 and Trp623(FIG. 1D) in generating compounds made of N-substituted(paracyclohexyl)benzyl analogs (FIGS. 1B and C).

A paper by Fletcher et al. entitled, Disruption of TranscriptionallyActive Stat3 Dimers with Non-Phosphorylated, Salicylic Acid-Based SmallMolecules: Potent In vitro and Tumor Cell Activities, which reports onthe details of the design and synthesis of the series of S3I-201 analogsmay be used by the skilled for guidance in synthesizing the novelcompounds disclosed herein (41).

Inhibition of Stat3 DNA-Binding Activity.

S3I201 analogs derived per in silico structural optimization andmolecular modeling of the binding to the Stat3 SH2 domain weresynthesized and evaluated in Stat3 DNA-binding assay in vitro, aspreviously done (18). Nuclear extracts containing activated Stat3prepared from v-Src transformed mouse fibroblasts (NIH3T3/v-Src) thatharbor aberrant Stat3 were incubated for 30 min at room temperature withor without increasing concentrations of the analogs, S3I201.1066 andS3I-201.2096, prior to incubation with the radiolabeled hSIE probe thatbinds to Stat3 and Stat1 and subjecting to electrophoretic mobilityshift assay (EMSA) analysis (18). Stat3 DNA-binding activity wasinhibited in a dose-dependent manner by both S3I-201.1066 andS3I201.2096 (FIGS. 2A(i) and (ii)), with average IC₅₀ values of 35±09 μMand 45±12 μM, respectively. These values represent 2-3 fold improvementover the activity of the lead agent, S3I-201 (IC₅₀ of 86 μM) (18), fromwhich the present compounds were derived. For selectivity, nuclearextracts containing activated Stat1, Stat3 and Stat5 prepared fromEGF-stimulated NIH3T3/hEGFR (mouse fibroblasts over-expressing the humanepidermal growth factor receptor, EGFR) were pre-incubated at roomtemperature with or without increasing concentrations of S3I-201.1066for 30 min, prior to incubation with the radiolabeled oligonucleotideprobes and subjecting to EMSA analyses, as previously done (18). EMSAresults of the binding studies using the hSIE probe shows the strongestcomplex of Stat3:Stat3 with the probe (upper band, lanes 1 and 2), whichis significantly disrupted at 50 μM S3I-201.1066 and completelydisrupted at 100 μM S3I-201.1066 (FIG. 2A(iii), upper band, lanes 2 and3). EMSA analysis further shows a less intense Stat1:Stat3 complex(intermediate band), which is similarly repressed at 50 μM andcompletely disrupted at 100 μM S3I-201.1066 (FIG. 2A(iii), lanes 2 and3). By contrast, we observe no significant inhibition of the Stat1:Stat1complex that is of the lowest intensity (lower band) at 50 μMS3I-201.1066, a moderate inhibition at 100 μM S3I-201.1066, while acomplete inhibition occurred at 200 μM S3I-201.1066 (FIG. 2A(iii), lowerband). Of importance, at the 100 μM S3I-201.1066 concentration at whichonly a moderate inhibition of Stat1:Stat1 complex occurred, the largerStat3:Stat3 complex is completely dissociated (FIG. 2A, lane 3).Moreover, EMSA analysis showed no effect on Stat5:Stat5 complex with theMGFe probe, up to 300 μM S3I-201.1066 (FIG. 2A(iv)). Thus, S3I-201.1066preferentially inhibits DNA-binding activity of Stat3 over that of Stat1and Stat5.

Inhibition of Intracellular Stat3 Activation.

Stat3 is constitutively activated in a variety of malignant cells,including human breast and pancreatic cancer cells (9,10,20). Given theeffect against Stat3 DNA-binding activity in vitro, we evaluatedS3I-201.1066 in v-Src transformed mouse fibroblasts (NIH3T3/v-Src),human breast cancer (MDA-MB-231) and human pancreatic cancer (Panc-1)lines that harbor aberrant Stat3 activity.

Twenty-four hours after treatment, nuclear extracts were prepared fromcells and subjected to Stat3 DNA-binding assay in vitro using theradiolabeled hSIE probe and analyzed by EMSA. Compared to the control(0.05% DMSO-treated cells, lane 1), nuclear extracts fromS3I-201.1066-treated NIH3T3/v-Src, Panc-1 and MDA-MB-231 cells showeddose-dependent decreases of constitutive Stat3 activation, withsignificant inhibition at 50 μM S3I-201.1066 (FIG. 2B, compare lanes2-5, 810, and 13-15 to their respective controls (0)). Luciferasereporter studies were performed to further determine the effect ofS3I-201.1066 on Stat3 transcriptional activity.

Results show that treatment with S3I-201.1066 of the v-Src transformedmouse fibroblasts (NIH3T3/v-Src) that stably express the Stat3-dependentluciferase reporter (pLucTKS3) (33) significantly (p<0.01) repressed theinduction of the Stat3-dependent reporter (FIG. 2C). SDS-PAGE andWestern blot analysis further showed that treatment with S3I201.1066 for24 h induced a concentration-dependent reduction of pTyr705Stat3 levelsin NIH3T3/v-Src (FIG. 2C(i), top panel) and Panc-1 cells (FIG. 2C(ii),top panel), presumably through the blockade of Stat3 binding to pTyrmotifs of receptors and the prevention of de novo phosphorylation bytyrosine kinases.

By contrast, immunoblotting analysis of whole-cell lysates from the twocell line models showed no significant effects of S3I-201.1066 on thephosphorylation of Src (pY416Src), Jak1 (pJak), Shc (pShc), and Erk1/2(pErk1/2) under the same treatment conditions, (FIGS. 2C (i) and (ii),panels 2-5 from the top), except the disappearance of pJak1 level inPanc-1 cells at 100 μM S3I201.1066. Total Stat3, Src, Jak1, Shc andErk1/2 protein levels remained unchanged. We infer that at theconcentrations that inhibit Stat3 activity, S3I-201.1066 has minimaleffect on Src, Jak1, Shc and Erk1/2 activation.

In Vitro Evidence that S3I-201.1066 Interacts with Stat3 (or SH2 Domain)and Selectively Disrupts Stat3 Binding to Cognate pTyr Peptide Motif ofReceptor.

Given the computational modeling prediction that S3I-201.1066 interactswith the Stat3 SH2 domain, we deduce that S3I201.1066 blocks Stat3DNA-binding activity by binding to the Stat3 SH2 domain, therebydisrupting Stat3:Stat3 dimerization. To determine therefore if the Stat3SH2 domain could interact with S3I-201.1066, we tested whether theaddition of the recombinant Stat3 SH2 domain into the DNA-binding assaymixture could intercept the inhibitory effect of the agent on Stat3activity that is observed in FIG. 2A(i). Purified histidine-tagged Stat3SH2 domain (His-SH2) was added at increasing concentrations (1-500 ng)to nuclear extracts containing activated Stat3 and the mixed extractswere subjected to DNA-binding assay in vitro for the study of the effectof S3I-201.1066, as was done in FIG. 2A(i). EMSA analysis shows a stronginhibition by S3I-201.1066 of Stat3 DNA-binding activity, as shown inFIG. 2A(i), when no His-SH2 domain was added to the nuclear extracts(FIG. 3A, lanes 2, 7, and 9 compared to lane 1). By contrast, theobserved S3I-201.0166-mediated inhibition of Stat3 DNA-binding activitywas progressively eliminated by the presence of an increasingconcentration of purified His-SH2 domain (Stat3 SH2), leading to thefull recovery of Stat3 activity when recombinant SH2 domain protein waspresent at 125-500 ng (FIG. 3A, lanes 3-6, 8 and 10).

The preceding studies suggest that S3I-201.1066 interacts with the Stat3SH2 domain. However, the studies do not demonstrate a direct binding tothe Stat3 SH2 domain. To provide definitive evidence of direct bindingto Stat3, biophysical studies were performed. His-tagged Stat3 protein(or SH2 domain; 50 ng) was immobilized on a Ni-NTA sensor chip surfacefor Surface Plasmon Resonance analysis of the binding of S3I201.1066 asthe analyte. Association and dissociation measurements were taken andthe binding affinity of S3I-201.1066 for Stat3 was determined using Qdatsoftware. Results showed a gradual increase and decrease with time inthe signals (response unit (RU)) for the association and dissociation,respectively, of the agent upon its addition to the immobilizedHis-Stat3 (FIG. 3B(iii)), indicative of the binding of S3I-201.1066 toand dissociation from the Stat3 protein. The curves depictedinteractions between Stat3 and S3I-201.1066, with a binding affinity,K_(D) of 2.74 nM/providing the first definitive evidence of direct Stat3binding for S3I-201 or its derivatives. The interactions also showed adependency on the concentration of the S3I-201.1066 (FIG. 3B(iii)). ThisSPR analysis of the conformational changes in His-Stat3 was validated byusing the high affinity Stat3-binding phosphoTyr (pY) peptide,GpYLPQTV-NH2 derived from the interleukin-6 receptor (IL-6R) subunit,gp-130 (22,23) (with a K_(D) of 24 nM) (FIG. 3B(ii)), and itsnon-phosphorylated counterpart, GYLPQTV-NH₂, which showed littleevidence of significant binding to Stat3 (FIG. 3B(i)). Interestingly,the dissociation curve for S3I-201.1066 showed a large residual bindingto Stat3 between 500-1000 s (FIG. 3B(iii), 10-50 μM, 500-1000 s),compared to the rapid association and dissociation of the high affinitypeptide to and from Stat3 with no residual binding of the phosphopeptide(FIG. 3B(ii)). The implication of this finding is presently unknown, butmay suggest a slower “off” rate for the dissociation of S3I-201.1066from Stat3. Differences in the chemical compositions and physicochemicalproperties would account for these different behaviors of interactionswith the Stat3 protein.

The studies so far demonstrate that S3I201.1066 interacts with Stat3 orthe Stat3 SH2 domain (data not shown). The interaction with the Stat3SH2 domain could block the binding of Stat3 to its cognate pTyr peptidemotifs of receptors. To verify that the agent disrupts pTyr-Stat3 SH2domain interactions, hence Stat3:Stat3 dimerization, we set up afluorescence polarization (FP) study based on the binding of Stat3 tothe high affinity peptide, GpYLPQTV-NH2 (22,23). It has previously beenreported that Stat3 binds to GpYLPQTV-NH2 with a higher affinity than tothe Stat3-derived pTyr peptide, PpYLKTK. This high affinity peptidedisrupted Stat3 DNA-binding activity in vitro with an IC₅₀ value of 0.15μM (22). The FP assay utilizing the 5carboxyfluorescein-GpYLPQTV-NH2 asa probe showed a saturation curve in the fluorescence polarizationsignal (mP) with increasing concentration (in μM) of purified His-Stat3for a robust Z′ value of 0.84 (FIG. 3C(i)), which closely matches thepreviously reported value of 0.87 (23).

Test of the non-phosphorylated, non-labeled GYLPQTV-NH2 in the FP assayshowed no evidence of effect on the fluorescent polarization signal(data not shown), while as expected, the phosphorylated, unlabeledcounterpart, GpYLPQTV-NH2 induced a complete inhibition with an IC₅₀value of 0.3 μM (data not shown), consistent with the previouslyreported value of 0.25±0.03 μM (23). The FP assay was used to furthertest the computational modeling prediction of the ability ofS3I-201.1066 to disrupt Stat3 interaction with its cognate pTyr peptide.Results show that S3I-201.1066, in a concentration-dependent manner,abrogated the fluorescent polarization signal for the interactionbetween the fluorescently-labeled phosphopeptide and Stat3 (FIG.3C(ii)). The inhibitory constant (IC₅₀ value) was derived to be 20±7.3μM, which is within the range for the IC₅₀ value (35±9 μM) determinedfor the inhibition of Stat3 DNA-binding activity (FIG. 2A(i)). Thesefindings together indicate that S3I-201.1066 binds to Stat3 or its SH2domain and disrupts the interaction of Stat3 with its pTyr peptide,thereby blocking Stat3 DNA-binding activity.

To further verify that S3I-201.1066 disrupts the binding of Stat3 toreceptors, mouse fibroblasts over-expressing the EGF receptor(NIH3T3/hEGFR) were treated with or without the compound prior tostimulation with EGF for 10 min. Cells were then subjected toimmunostaining for EGFR (red) and Stat3 (green) and analyzed by confocalmicroscopy for EGF-induced colocalization of Stat3 and EGFR and forStat3 nuclear translocation, as previously performed (17). In theresting NIH3T3/hEGFR fibroblasts, EGFR (red) is widely localized at theplasma membrane, while Stat3 (green) is localized at both the plasmamembrane and in the cytoplasm, with a minimal colocalization with EGFRat the plasma membrane and no visible presence in the nucleus (stainedblue for DAPI) (FIG. 4A upper panel). Stimulation by EGF of cellsuntreated with S3I-201.1066 induced a strong nuclear presence of Stat3(cyan for merged Stat3 (green) and DAPI (blue)), as well ascolocalizations of EGFR and Stat3 (yellow for merged EGFR (red) andStat3 (green)) at the plasma membrane, cytoplasm, and peri-nuclear space(FIG. 4A, bottom left). This EGF-stimulated colocalization between EGFRand Stat3 and the Stat3 nuclear localization were both strongly blockedwhen cells were pre-treated with S3I-201.1066 before stimulating withEGF (FIG. 4A, bottom right compared to non-treated, bottom left),indicating that the compound disrupts Stat3 binding to EGFR. We inferthat by blocking Stat3 binding to EGFR, S3I-201.1066 attenuates Stat3phosphorylation/activation and thereby prevents Stat3 nucleartranslocation. To investigate further the Stat3 interaction with theEGFR receptor and the effect of S3I-201.1066, coimmunoprecipitation withimmunoblotting studies were performed in which EGFR immunecomplex wasimmunoprecipitated from whole-cell lysates prepared from treated anduntreated cancer cells and blotted for Stat3, and for Shc and Grb 2 asnegative control. Results showed that EGFR immunecomplex precipitatedfrom untreated Panc1 and MDA-MB-231 cells contained Stat3, Shc and Grb 2(FIG. 4B(i), lanes 1 and 3, i.p. EGFR, blot Stat3, Shc, and Grb 2). Bycontrast, treatment of both cell lines with S3I-201.1066 significantlydiminished the level of Stat3 that associated with EGFR in theimmunecomplex of equal total protein without affecting the Shc or Grb 2levels that are associated with EGFR in the complex. See FIG. 4B(i),lanes 2 and 4, i.p. EGFR, blot Stat3, Shc and Grb 2. Western blotting ofwhole-cell lysates of equal total protein shows the levels of activatedand total Erk1/2 are unaffected by the treatment of cells withS3I-201.1066 (FIG. 4B(i), input, blot pErk and Erk) and the Stat3protein levels remain the same (FIG. 4B(i), input, blot Stat3).

In other studies, EGFR and Stat3 immunecomplexes were independentlyprecipitated from whole-cell lysates of untreated Panc-1 cells andcomplexes of equal total protein were directly treated with 0-100 MS3I-201.1066 for 3 h and then subjected to Western blotting analysis.Compared to untreated samples (FIG. 4B(ii), lane 1), results show thatthe direct treatment with S3I-201.1066 of the EGFR immunecomplexdramatically diminished the level of Stat3 present in the complex (FIG.4B(ii), i.p. EGFR, blot Stat3, lanes 2-4), with no visible changes inthe levels of Shc or Grb 2 present in the complex (FIG. 4B(ii), i.p.,EGFR, blot Shc or Grb 2). The EGFR levels in the immunecomplexesremained unchanged (FIG. 4B(ii), upper band). Similarly, the Stat3immunecomplex that is directly treated with S3I-201.1066 and blotted forEGFR showed strongly reduced EGFR levels, compared to the untreatedStat3 immunecomplex of equal total protein (FIG. 4B(ii), i.p. Stat3,blot EGFR, compare lane 1 to lanes 2-4). The Stat3 levels in theimmunecomplexes remained unchanged (FIG. 4B(ii), i.p. Stat3, blotStat3). Altogether, these findings strongly indicate that S3I-201.1066disrupts the binding of Stat3 to cognate receptor motifs, therebyblocking Stat3 phosphorylation and nuclear translocation.

S3I-201.1066 Blocks Growth, Viability, Malignant Transformation, and theMigration of Cells Harboring Constitutively-Active Stat3.

Aberrant Stat3 promotes malignant cell proliferation, survival andmalignant transformation (10,20,42). Given that S3I201.1066 disruptsStat3 activation, we asked the question whether this agent is able toselectively decrease the viability and growth of malignant cells thatharbor aberrant Stat3 activity. The human breast (MDA-MB-231) andpancreatic cancer (Panc-1) lines and the v-Src-transformed mousefibroblasts (NIH3T3/v-Src) that harbor constitutively-active Stat3, aswell as cells that do not harbor aberrant Stat3 (normal mousefibroblasts (NIH3T3), v-Ras-transformed counterpart fibroblasts(NIH3T3/v-Ras), Stat3 knockout mouse embryonic fibroblasts (MEFs)(Stat3−/−) (31), normal human pancreatic duct epithelial cells (HPDEC)(30), and mouse thymic epithelial stromal cells (TE-71) cells (32)) inculture were treated with or without an increasing concentration ofS3I-201.1066 for up to 6 days and analyzed for viable cell numbers byCyQuant cell proliferation/viability kit (FIG. 5A) and by trypan blueexclusion with phase-contrast microscopy (FIG. 5B), as described in the“Materials and Methods”.

Compared to the control (DMSO-treated) cells, the mouse fibroblaststransformed by v-Src (NIH3T3/v-Src), and the MDA-MB-231 and Panc-1 cellsshowed significantly reduced viable cell numbers (FIG. 5A) and weregrowth inhibited (FIG. 5B(ii)-(iv)) following treatment with increasingconcentrations of S3I-201.1066 for 24-144 h. By contrast, the viability(FIG. 5A) and growth (FIG. 5B(i) and (v)) of NIH3T3, Stat3-null MEFs(Stat3−/−), normal human pancreatic duct epithelial cells (HPDEC), andthe mouse thymus epithelial stromal cells (TE71) that do harbor aberrantStat3 activity were not significantly altered by up to 200 μMS3I201.1066 treatment (FIG. 5A and B, and data not shown), with derivedIC₅₀ values that are >200 μM, compared to 35, 48, and 37 μM forNIH3T3/v-Src, Panc-1, and MDA-MB-231, respectively (FIG. 5A, lowerpanel). These findings suggest that S3I-201.1066 exerts preferentialbiological effects against malignant cells that harborconstitutively-active Stat3, and at concentrations that inhibit Stat3activity, the agent does not affect other cells.

We extended these studies to examine the effect of S3I-201.1066 incolony survival assay performed as previously reported (39). Culturedsingle-cells were untreated or treated once with S3I-201.1066 andallowed to grow until large colonies were visible, which were stainedand enumerated.

Results showed a dose-dependent suppression of the number of coloniesfor the v-Src transformed mouse fibroblasts (NIH3T3/v-Src), the humanpancreatic cancer, Panc-1 and the human breast cancer, MDA-MB-231 cells(FIG. 5C(ii)-(iv)). By contrast, minimal effect was observed on thecolony numbers for mouse fibroblasts transformed by v-Ras (NIH3T3/v-Ras)that do not harbor constitutively-active Stat3 (FIG. 5C(i)).Furthermore, growth in soft-agar suspension of NIH3T3/v-Src, MDA-MB-231and Panc-1 cells treated with S3I-201.1066 was significantly inhibited(FIG. 6A(ii)-(iv)). By comparison, at concentrations that inhibit Stat3,S3I-201.1066 showed minimal effect on the soft-agar growth of v-Rastransformed mouse fibroblasts (NIH3T3/v-Ras) (FIG. 6A(i)). Thesefindings indicate that S3I-201.1066 selectively suppresses viability,growth, and survival of malignant cells harboring aberrant Stat3, andblocks Stat3-mediated malignant transformation. Thus, these studiesdemonstrate that Stat3 is important not only for tumor growth, but alsotumor progression (43,44).

To further investigate the biological effects of S3I-201.1066 and toassess the ability to block Stat3-dependent tumor progression processes,a wound healing study was performed, as described in “Materials andMethods” section for monitoring the migration of malignant cells and theeffect of treatment with S3I-201.1066. Significantly reduced numbers ofMDA-MB-231, Panc-1 and NIH3T3/v-Src cells migrating into the denudedarea were observed following 12-24 h treatment with S3I-201.1066 (FIG.6B and data not shown), with strongly reduced numbers occurring at 50 or100 μM S3I201.1066 treatment, and statistically lower numbers at 30 MS3I-201.1066 (FIG. 6B). By contrast, the migration of NIH3T3/v-Rasfibroblasts was minimally affected by the same treatment conditions(FIG. 6B). In the 12-24 h treatment duration, there was no evidence ofapoptosis of the treated cells (data not shown). These findingsdemonstrate that S3I-201.1066 selectively suppresses the migration ofmalignant cells that harbor aberrant Stat3.

S3I-201.1066 Represses the Expression of c-Myc, Bcl-xL, VEGF, and MMP-9.

Known Stat3 downstream target genes are key in the dysregulatedbiological processes promoted by aberrant Stat3 (9,20,42). We sought tovalidate the inhibitory effect of S3I-201.1066 on aberrant Stat3signaling and to define the underlying molecular mechanisms for theantitumor cell effects of the agent by investigating changes in theinduction of known Stat3-regulated genes. In the human breast carcinoma,MDA-MB-231 and pancreatic cancer, Panc-1 cell lines that harborconstitutively-active Stat3, immunoblotting analysis of whole-celllysates shows the constitutive induction of known Stat3-regulated genes,including c-Myc, Bcl-xL, VEGF, and MMP-9 proteins, which weresignificantly suppressed in response to 48 h-treatment with S3I-201.1066(FIG. 7 and data not shown). These data indicate that S3I-201.1066sufficiently represses the constitutive induction of Stat3-regulatedgenes, thereby thwarting the effect of aberrant Stat3 in terms ofeliminating the dysregulation of growth and survival that supports themalignant phenotype. The ability of S3I-201.1066 to block Stat3transcriptional activity is also supported by the data in FIG. 2C.

S3I-201.1066 Inhibits Growth of Human Breast Tumor Xenografts.

Given Stat3's importance in tumor growth and tumor progression, weevaluated S3I-201.1066 in xenograft models of the human breast cancer(MDA-MB-231) cells that harbor aberrant Stat3. Compared to control(vehicle-treated), tumor-bearing mice, treated (i.v. injection) withS3I-201.1066 at 3 mg/kg every 2 or 3 days for 17 days had greatlyreduced tumor sizes (FIG. 8). Animals remained viable at this dose andshowed no obvious sign of toxicity. These findings together demonstratethat S3I-201.1066 induces antitumor cell effects and tumor regression bytargeting the Stat3 SH2 domain and thereby inhibiting Stat3-mediatedtumor processes.

Discussion

Computational modeling of the interactions of the Stat3 SH2 domain withthe previously reported Stat3 inhibitor lead, S3I-201 (18) derived keystructural information for optimization and a rational synthetic programthat furnished exciting new analogs. The compounds disclosed herein,S3I-201.1066 (Formula 1) and S3I-201.2096 (Formula 2) show improvedStat3-inhibitory inhibitory potency and selectivity in vitro, withintracellular Stat3-inhibitory activity that is enhanced 2-3-fold.Analog S3I-201.1066 exhibited an improved target selectivity and showeda minimum inhibitory effect on the phosphorylation of Src, Jak1,Erk1/2MAPK and Shc proteins at concentrations (30-50 μM) that inhibitintracellular Stat3 activation, despite there being SH2 domains involvedin the mechanisms leading to the activation of these other proteins. Permolecular modeling, the improved activity could in part be due to theenhanced interactions with the Stat3 protein, possibly by the (paracyclohexyl)benzyl moiety that extends from the scaffold amide nitrogenand makes important contacts with the hydrophobic residuesTrp623,11e659, Val637 and Phe716 within the unexplored pocket.

The native Stat3 peptide inhibitor, PpYLKTK (where pY represents pTyr)and its peptidomimetic analogs (15,16) and several other Stat3 SH2domain-binding and dimerization disrupting peptides and theirderivatives have been reported (21,22,25). Previous studies haveutilized the fluorescence polarization analysis to characterize thebinding of the native, high affinity phosphopeptide, GpYLPQTV-NH2 (as5carboxyfluorescein-GpYLPQTV-NH2) to the Stat3 protein (22,23). Usingthis assay platform and surface plasmon resonance analysis, we providedefinitive evidence for the physical interaction of S3I-201.1066 withStat3 or its SH2 domain, with an affinity (KD) of 2.3 μM.

The analysis of the interaction reveals a slower kinetics of theassociation and dissociation, which contrasts the more rapid binding anddissociation of the native, high affinity peptide, GpYLPQTV-NH2 to andfrom Stat3, with a corresponding affinity (KD) of 24 nM. The implicationof these differences in the binding kinetics in relation to themodulation of Stat3 function is presently unclear.

The second supporting evidence for the interaction of S3I201.1066 withStat3 comes by way of the disruption by S3I-201.1066 of the Stat3binding to the pTyr peptide in a fluorescent polarization assay based onthe high affinity peptide, 5carboxyfluorescein-GpYLPQTV-NH2 probe andStat3, with a derived IC₅₀ of 45 μM.

By comparison, the unlabeled, native phosphopeptide disrupts thisinteraction between the probe and Stat3, with an IC₅₀ value of 0.3 μM,which is in line with similar reported studies of the high affinitypeptide (22,23) that derived an affinity of 0.15±0.01 μM (23) and anIC50 value of 0.290±0.063 μM (21). The higher affinity of the nativepeptide for the protein should be expected, given the more favorablephysicochemical properties that will facilitate a stronger binding tothe Stat3 protein.

Overall, our study provides support for the binding of S3I-201.1066 toStat3, and for its ability to disrupt the interaction between Stat3 andits cognate pTyr peptide, an event that is indicative of Stat3:Stat3dimerization. Although other Stat3 dimerization disruptors have beenpreviously identified through molecular modeling (19,45), the presentstudy is the first to provide biophysical evidence for a directinteraction of a small-molecule, dimerization disruptor with the Stat3protein. Given the disruption of the Stat3 binding to the cognatepeptide, GpYLPQTV-NH2, we infer that inside cells, S3I-201.1066 mayinterfere with the ability of Stat3 (via SH2 domain) to bind to cognatepTyr motifs on receptors and thereby block de novo phosphorylation bytyrosine kinases, as well as disrupt pre-existing Stat3:Stat3 dimers,particularly in malignant cells that harbor aberrant Stat3. Indeed, ourstudy shows a strong association of Stat3 with EGFR in ligand-stimulatedmouse fibroblasts or in cancer cells, and a strong presence in thenucleus of stimulated cells. Both the Stat3:EGFR association and theStat3 nuclear presence are blocked by S31201.1066, indicating that bydisrupting Stat3 binding to receptors, S3I-201.1066 prevents Stat3phosphorylation, activation, and nuclear translocation, therebyattenuating Stat3 function.

Substantive evidence demonstrates that aberrant Stat3 activity promotescancer cell growth and survival (15,16,29,46,47), and induces tumorangiogenesis (48,49) and metastasis (43,49). Accordingly, inhibitors ofStat3 activation and signaling have been shown to induce antitumor celleffects consistent with the abrogation of Stat3 function(15-19,37,50-52).

The present disclosure parallels these published reports in showing thatS3I-201.1066 induces growth inhibition and loss of viability andsurvival of the human pancreatic cancer Panc-1 and breast cancerMDA-MB-231 cells, and of the v-Src transformed mouse fibroblasts(NIH3T3/v-Src), which are restricted to malignant cells that harboraberrant Stat3, while the effects on normal human pancreatic ductepithelial cells, normal mouse fibroblasts, mouse thymic epithelialstromal cells, the viral Ras-transformed mouse fibroblasts that do notharbor aberrant Stat3, and the Stat3 knockout mouse embryonicfibroblasts (Stat3−/−) (31) are minimal.

Moreover, S3I-201.1066-induced antitumor cell effects occurred atsignificantly lower concentrations, 30-50 μM, than the 100 μM activitypreviously reported for the lead agent (18). Mechanistic insight intothe biological effects of S3I-201.1066 as a Stat3 inhibitor is providedby the evidence of a suppression of the constitutive expression of knownStat3-regulated genes, including c-Myc, Bcl-xL, VEGF and MMP-9, and thedisruption of the Stat3 binding to receptor, which control cell growthand apoptosis, promote tumor angiogenesis, or modulate invasion(19,43,46,49,53,54). The exclusion of Stat3 from the nucleus furthercontributes to the inhibition of Stat3 transcriptional function. Wefurther note the significant antitumor effect of S3I-201.1066 in humanbreast tumor xenografts. Altogether the present disclosure providesevidence for the binding of S3I-201.1066 to Stat3, disruption ofStat3:pTyr interactions and hence Stat3:Stat3 dimerization, and thedisruption of the Stat3 binding to receptor, phosphorylation and nucleartranslocation.

Accordingly, in the drawings and specification there have been disclosedtypical preferred embodiments of the invention and although specificterms may have been employed, the terms are used in a descriptive senseonly and not for purposes of limitation. The invention has beendescribed in considerable detail with specific reference to theseillustrated embodiments. It will be apparent, however, that variousmodifications and changes can be made within the spirit and scope of theinvention as described in the foregoing specification and as defined inthe appended claims.

REFERENCES CITED

-   1. Bromberg, J. (2000) Breast Cancer Res. 2, 86-90.-   2. Darnell, J. E., Jr. (2002) Nat. Rev. Cancer 2, 740-749.-   3. Schroder, M., Kroeger, K., Volk, H. D., Eidne, K. A., and    Grutz, G. (2004) J. Leukoc Biol. 75, 792-797.-   4. Sehgal, P. B. (2008) Dev Biol 19 329-340.-   5. Bromberg, J., and Darnell, J. E., Jr. (2000) Oncogene 19,    2468-2473.-   6. Bowman, T., Garcia, R., Turkson, J., and Jove, R. (2000) Oncogene    19, 2474-2488.-   7. Turkson, J., and Jove, R. (2000) Oncogene 19, 6613-6626.-   8. Buettner, R., Mora, L. B., and Jove, R. (2002) Clin. Cancer Res.    8, 945-954.-   9. Yu, H., and Jove, R. (2004) Nat. Rev. Cancer 4, 97-105.-   10. Turkson, J. (2004) Expert Opin Ther Targets 8, 409-422.-   11. Darnell, J. E. (2005) Nat Med. 11, 595-596.-   12. Kortylewski, M., and Yu, H. (2007) J Immunother. 30, 131-139.-   13. Kortylewski, M., and Yu, H. (2008) Curr Opin Immunol. 20,    228-233.-   14. Shuai, K., Horvath, C. M., Huang, L. H., Qureshi, S. A.,    Cowburn, D., and Darnell, J. E., Jr. (1994) Cell 76, 821-828.-   15. Turkson, J., Ryan, D., Kim, J. S., Zhang, Y., Chen, Z., Haura,    E., Laudano, A., Sebti, S., Hamilton, A. D., and Jove, R. (2001) J.    Biol. Chem. 276, 45443-45455.-   16. Turkson, J., Kim, J. S., Zhang, S., Yuan, J., Huang, M., Glenn,    M., Haura, E., Sebti, S., Hamilton, A. D., and Jove, R. (2004) Mol    Cancer Ther 3, 261-269.-   17. Siddiquee, K., Glenn, M., Gunning, P., Katt, W. P., Zhang, S.,    Schroeck, C., Jove, R., Sebti, S., Hamilton, A. D., and    Turkson, J. (2007) ACS Chem Biol. 2 787-798.-   18. Siddiquee, K., Zhang, S., Guida, W. C., Blaskovich, M. A.,    Greedy; B., Lawrence, H., Yip, M. L. R., Jove, R., McLaughlin, M.,    Lawrence, N., Sebti, S., and Turkson, J. (2007) 1: Proc Natl Acad    Sci USA. 104 7391-7396.-   19. Song, H., Wang, R., Wang, S., and Lin, J. (2005) Proc Natl Acad    Sci U S A. 102, 47004705.-   20. Yue, P., and Turkson, J. (2009) Expert Opin Investig Drugs. 18    45-56.-   21. Coleman, D. R. I., Ren, Z., Mandal, P. K., Cameron, A. G.,    Dyer, G. A., Muranjan, S., Campbell, M., Chen, X., and    McMurray, J. S. (2005) J. Med. Chem. 48, 6661-6670.-   22. Ren, Z., Cabell, L. A., Schaefer, T. S., and    McMurray, J. S. (2003) Bioorg Med Chem Lett 13, 633-636.-   23. Schust, J., and Berg, T. (2004) Anal. Biochem. 330 114-118.-   24. Gunning, P. T., Glenn, M. P., Siddiquee, K. A., Katt, W. P.,    Masson, E., Sebti, S. M., Turkson, J., and Hamilton, A. D. (2008)    Chembiochem. 9, 2800-2803.-   25. Fletcher, S., Turkson, J., and Gunning, P. T. (2008) ChemMedChem    3, 1159-1168 .-   26. Becker, S., Groner, B., and Muller, C. W. (1998) Nature 394,    145-151.-   27. Johnson, P. J., Coussens, P. M., Danko, A. V., and    Shalloway, D. (1985) Mol. Cell. Biol. 5, 1073-1083.-   28. Yu, C. L., Meyer, D. J., Campbell, G. S., Lamer, A. C.,    Carter-Su, C., Schwartz, J., and Jove, R. (1995) Science 269, 81-83.-   29. Garcia, R., Bowman, T. L., Niu, G., Yu, H., Minton, S.,    Muro-Cacho, C. A., Cox, C. E., Falcone, R., Fairclough, R., Parson,    S., Laudano, A., Gazit, A., Levitzki, A., Kraker, A., and    Jove, R. (2001) Oncogene 20, 2499-2513.-   30. Ouyang, H., Mou, L. J., Luk, C., Liu, N., Karaskova, J., Squire,    J., and Tsao, M. S. (2000) Am. J. Pathol. 157, 1623-1631.-   31. Maritano, D., Sugrue, M. L., Tininini, S., Dewilde, S., Strobl,    B., Fu, X., Murray-Tait, V., Chiarle, R., and Poli, V. (2004) Nat    Immunol. 5, 401-409.-   32. Farr, A. G., Hosier, S., Braddy, S. C., Anderson, S. K.,    Eisenhardt, D. J., Yan, Z. J., and Robles, C. P. (1989) Cell    Immunol. 119, 427-444.-   33. Turkson, J., Bowman, T., Garcia, R., Caldenhoven, E., De    Groot, R. P., and Jove, R. (1998) Mol. Cell. Biol. 18, 2545-2552.-   34. Wagner, M., Kleeff, J., Friess, H., Buchler, M. W., and    Korc, M. (1999) Pancreas. 19, 370-376.-   35. Gouilleux, F., Moritz, D., Humar, M., Moriggl, R., Berchtold,    S., and Groner, B. (1995) Endocrinology 136, 5700-5708.-   36. Seidel, H. M., Milocco, L. H., Lamb, P., Darnell, J. E., Jr.,    Stein, R. B., and Rosen, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92,    3041-3045.-   37. Turkson, J., Zhang, S., Mora, L. B., Burns, A., Sebti, S., and    Jove, R. (2005) J Biol Chem. 280, 32979-32988.-   38. Zhang, Y., Turkson, J., Carter-Su, C., Smithgall, T., Levitzki,    A., Kraker, A., Krolewski, J. J., Medveczky, P., and    Jove, R. (2000) J. Biol. Chem. 275, 24935-24944.-   39. Zhao, S., Venkatasubbarao, K., Lazor, J. W., Sperry, J., Jin,    C., Cao, L., and Freeman, J. W. (2008) Cancer Res 68, 4221-4228.-   40. Jones, G., Willett, P., Glen, R. C., Leach, A. R., and    Taylor, R. (1997) J. Mol. Biol. 267, 727-748.-   41. Fletcher, S., Jardeephi, S., Zhang, X., Yue, P., Page, B. D.,    Sharmeen, S., Shahani, V., Schimmer, A., Turkson, J., and    Gunning, P. T. (2009) ChemBioChem 10, 1959-1964.-   42. Siddiquee, K. A. Z. and Turkson, J. (2008) Cell Res. 18,    254-267.-   43. Xie, T. X., Wei, D., Liu, M., Gao, A. C., Ali-Osman, F., Sawaya,    R., and Huang, S. (2004) Oncogene 23, 3550-3560.-   44. Huang, C., Cao, J., Huang, K. J., Zhang, F., Jiang, T., Zhu, L.,    and Qiu, Z. J. (2006) Cancer Sci 97, 1417-1423.-   45. Bhasin, D., Cisek, K., Pandharkar, T., Regan, N., Li, C.,    Pandit, B., Lin, J., and Li, P. (2008) Bioorg. Med. Chem. Lett. 18,    391-395.-   46. Catlett-Falcone, R., Landowski, T. H., Oshiro, M. M., Turkson,    J., Levitzki, A., Savino, R., Ciliberto, G., Moscinski, L.,    Fernandez-Luna, J. L., Nunez, G., Dalton, W. S., and Jove, R. (1999)    Immunity 10, 105-115.-   47. Mora, L. B., Buettner, R., Seigne, J., Diaz, J., Ahmad, N.,    Garcia, R., Bowman, T., Falcone, R., Fairclough, R., Cantor, A.,    Muro-Cacho, C., Livingston, S., Karras, J., Pow-Sang, J., and    Jove, R. (2002) Cancer Res 62, 6659-6666.-   48. Niu, G., Wright, K. L., Huang, M., Song, L., Haura, E., Turkson,    J., Zhang, S., Wang, T., Sinibaldi, D., Coppola, D., Heller, R.,    Ellis, L. M., Karras, J., Bromberg, J., Pardoll, D., Jove, R., and    Yu, H. (2002) Oncogene 21, 2000-2008.-   49. Wei, D., Le, X., Zheng, L., Wang, L., Frey, J. A., Gao, A. C.,    Peng, Z., Huang, S., Xiong, H. Q., Abbruzzese, J. L., and    Xie, K. (2003) Oncogene 22, 319-329.-   50. Fuh, B., Sobo, M., Cen, L., Josiah, D., Hutzen, B., Cisek, K.,    Bhasin, D., Regan, N., Lin, L., Chan, C., Caldas, H., DeAngelis, S.,    Li, C., Li, P., and Lin, J. (2009) Br. J. Cancer 100, 106-112.-   51. Blaskovich, M. A., Sun, J., Cantor, A., Turkson, J., Jove, R.,    and Sebti, S. M. (2003) Cancer Res 63, 1270-1279.-   52. Sun, J., Blaskovich, M. A., Jove, R., Livingston, S. K.,    Coppola, D., and Sebti, S. M. (2005) Oncogene. 24, 3236-3245.-   53. Real, P. J., Sierra, A., De Juan, A., Segovia, J. C.,    Lopez-Vega, J. M., and Fernandez-Luna, J. L. (2002) Oncogene 21,    7611-7618.-   54. Gritsko T, Williams A, Turkson J, Kaneko S, Bowman T, Huang M,    Nam S, Eweis I, Diaz N, Sullivan D, Yoder S, Enkemann S, Eschrich S,    Lee J H, Beam C A, Cheng J, Minton S, Muro-Cacho C. A., and    Jove, R. (2006) Clin Cancer Res. 12, 11-19.

That which is claimed:
 1. A compound according to formula 1 and saltsthereof.


2. The compound of claim 1, used in a pharmaceutical compositionacceptable for administration to a patient.
 3. The compound of claim 1,used in a method of treatment effective to inhibit a cancer cell bycontacting the cell with said compound.
 4. The compound of claim 1, usedin a method of treatment effective to inhibit a human pancreatic cancercell by contacting the cell with said compound.
 5. The compound of claim1, used in a method of treatment effective to inhibit a human breastcancer cell by contacting the cell with said compound.
 6. The compoundof claim 1, used in a method of treatment effective to inhibit a cellcharacterized by an aberrant level of Stat3 by contacting the cell withsaid compound.
 7. The compound of claim 1, used to inhibit a cellcharacterized by an aberrant level of Stat3 by contacting the cell withsaid compound so as to selectively bind Stat3.
 8. The compound of claim1, used to down-regulate expression of Stat3-regulated genes in a cellby contacting the cell with said compound.
 9. The compound of claim 1,used to selectively inhibit Stat3-DNA binding activity in a cell bycontacting the cell with said compound.
 10. The compound of claim 1,used to block Stat3 association with epidermal growth factor receptor inEGF-stimulated fibroblasts by contacting the fibroblasts with saidcompound.
 11. The compound of claim 1, used to inhibit tumor cellsdependent on aberrant Stat3-mediated oncogenesis by contacting the tumorcells with said compound so as to interfere with Stat3 function.
 12. Acompound according to formula 2 and salts thereof.


13. The compound of claim 12, used in a pharmaceutical compositionacceptable for administration to a patient.
 14. The compound of claim12, used in a method of treatment effective to inhibit a cancer cell bycontacting the cell with said compound.
 15. The compound of claim 12,used in a method of treatment effective to inhibit a human pancreaticcancer cell by contacting the cell with said compound.
 16. The compoundof claim 12, used in a method of treatment effective to inhibit a humanbreast cancer cell by contacting the cell with said compound.
 17. Thecompound of claim 12, used in a method of treatment effective to inhibita cell characterized by an aberrant level of Stat3 by contacting thecell with said compound.
 18. The compound of claim 12, used to inhibit acell characterized by an aberrant level of Stat3 by contacting the cellwith said compound so as to selectively bind Stat3.
 19. The compound ofclaim 12, used to down-regulate expression of Stat3-regulated genes in acell by contacting the cell with said compound.
 20. The compound ofclaim 12, used to selectively inhibit Stat3-DNA binding activity in acell by contacting the cell with said compound.
 21. The compound ofclaim 12, used to block Stat3 association with epidermal growth factorreceptor in EGF-stimulated fibroblasts by contacting the fibroblastswith said compound.
 22. The compound of claim 12, used to inhibit tumorcells dependent on aberrant Stat3-mediated oncogenesis by contacting thetumor cells with said compound so as to interfere with Stat3 function.